Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica

Background Eicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level. Results This study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (GPAT) genes from Schizochytrium sp. HX-308 were expressed in Yarrowia lipolytica to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of DGATs through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs “HFS” in ScDGAT2A and “FFG” in ScDGAT2B with the motif “YFP”. Notably, combined with lipidomic analysis, the expression of ScDGAT2C and GPAT2 enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the ScACC, ScACS, ScPDC, and ScG6PD genes from Schizochytrium sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively. Conclusion This study shows that the expression of lipid synthesis genes from Schizochytrium sp. in Y. lipolytica effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-024-02471-y.

from deep-sea fish oil.However, the unsustainability of deep-sea fish resources and the severe damage to marine ecosystems due to overfishing have limited the production and sale of EPA [2].In addition, the enormous gap between global EPA production and recommended dietary intake indicates the need to develop a sustainable and safe method for producing EPA [3].Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids because they can accumulate lipids exceeding 20% of their dry cell weight (DCW) [4].In addition, they have several advantages such as ease of culture, multiple lipid composition, and extensive carbon source substrate utilization [5].
Triglyceride (TG) is the major storage lipid in oleaginous cells, which is formed mainly through the Kennedy pathway and exists in both the endoplasmic reticulum and the chloroplasts (Additional file 1: Fig. S1) [6].Diacylglycerol acyltransferase (DGAT ) is considered a rate-limiting and key enzyme that catalyzes the final acylation of diacylglycerol (DG) to form TG [7].Previous studies have shown when CrDGTT1 from Chromochloris zofingiensis was expressed in Nannochloropsis oceanica, the percentage of EPA in TG and the content of TG-derived EPA increased by 4.8-fold and 10.1-fold, respectively, compared to the control strain [8].There are three distinct types of DGAT s related to TG biosynthesis, including DGAT 1, 2, and 3. DGAT 1 and DGAT 2 are membrane-binding protein and DGAT 3 is soluble cytosolic enzyme [9].Although they catalyze the same enzymatic reaction, there is no apparent homology in the amino acid sequence between DGAT1 and DGAT2.Modification of DGAT genes has already been attempted and is considered to be the best strategy for manipulating TG accumulation in microorganisms [10].DGAT2 has been identified as a more efficient TG biosynthetic enzyme than DGAT1, especially for unusual fatty acids, and has been transferred to various cells to produce and store large amounts of fatty acids [11].The functions of four DGAT2s (DGAT2A, DGAT2B, DGAT2C, and DGAT2D) in Aurantiochytrium sp.SD116 were investigated, and it was found that DGAT2C was mainly responsible for connecting PUFAs to the sn-3 position of the TG molecules, while DGAT2A and DGAT2D mediated the SFA-TGs production [5].Overexpression of DGAT2 in Nannochloropsis oceanica resulted in a 69% increase in TG content.In addition, SFAs and PUFAs were found to be increased whereas monounsaturated fatty acids content decreased [12].In another study, overexpression of DGAT2 in Chlamydomonas reinhardtii resulted in a ninefold increase in TG content [13].Therefore, DGAT is considered a key target for manipulating TG synthesis, and a better understanding of DGAT properties may provide new possibilities for the enrichment of required fatty acids in TG.Schizochytrium sp., as a marine heterotrophic microorganism, can synthesize and accumulate large amounts of TG and is rich in PUFAs [14].Under optimized conditions, the lipid content of some Schizochytrium strains can reach over 55% of DCW [15].Therefore, the exploration of its TG and lipid synthesis pathway genes is of great research significance.In this study, Y. lipolytica was used as a chassis cell into which the TG and lipid synthesis genes from Schizochytrium sp.HX-308 were introduced to enrich EPA in its TG for the first time through a "push-pull-promote" metabolic engineering strategy.Meanwhile, we combined lipidomics techniques with protein engineering to explore their role in mediating EPA in TG assembly.This study aims to provide a valuable reference for the improvement of EPA biosynthesis.

Bioinformatic analysis of DGAT s
Four putative DGAT genes, ScDGAT2A (gene ID: A6553), ScDGAT2B (gene ID: A7082), ScDGAT2C (gene ID: A7100), and ScDGAT3 (gene ID: A7950), were identified based on functional annotation of the genome of Schizochytrium sp.HX-308.ID: A7950).Based on the phylogenetic analysis of amino acid sequences, ScD-GAT2A, ScDGAT2B, and ScDGAT2C were classified as DGAT2 family, while ScDGAT3 was classified as DGAT3 family (Additional file 1: Fig. S2a).It has been reported that the DGAT3 clade shares a most recent ancestor with a group of uncharacterized proteins from cyanobacteria, suggesting a prokaryotic origin, whereas the canonical DGAT s, DGAT1 and DGAT2, are only related to proteins from eukaryotes [16].In addition, most microalgae contain one or more DGAT genes, which may be closely related to their high-level storage of PUFAs.According to reports, the DHA content of Schizochytrium sp. can reach over 35% of total fatty acids [17].
Subcellular localization prediction of the proteins showed that all of ScDGAT2A, ScDGAT2B, ScDGAT2C, and ScDGAT3 are localized in the endoplasmic and participate in the conventional pathway of TG synthesis here (Additional file 1: Fig. S4) [19].In addition, ScDGAT3 was also found to be localized in chloroplasts.According to reports, most of the algal sequences within the DGAT3 group have predicted chloroplast localization [20], indicating that they may have the ability to accumulate lightdependent TG [16].However, Schizochytrium sp. is a type of marine microalgae that lacks chloroplasts.Therefore, further experimental verification on this speculation is needed.The conservative domain prediction showed that ScDGAT2A, ScDGAT2B, and ScDGAT2C include a lysophospholipid acyltransferase (LPLAT) superfamily domain, which may be involved in the acyltransferase activity (Additional file 1: Fig. S5).While ScDGAT3 includes the abhydrolase superfamily domain, which belongs to the α/β hydrolase superfamily that contains proteases, lipases, peroxidases, esterases, epoxide hydrolases and dehalogenases [21].

Effects of DGAT s on lipid and fatty acid profile
In order to characterize the functions of four DGAT genes, yeast expression vectors for these genes were constructed and integrated into the intF3 loci of the Y. lipolytica mutant strain (CK) genome (Fig. 1a), in which DGA1, DGA2, and LRO1 that catalyze TG formation were all knocked out, resulting in fatty acids that can only exist in free form [22].In addition, the intF3 loci were selected based on previous reports that they can be used to integrate heterologous genes without affecting cell growth [23].Subsequently, the genomic DNA of Y. lipolytica was extracted and used as a template for PCR amplification, and electrophoretic bands of the target size were successfully obtained (Additional file 1: Fig. S6a), indicating that these DGAT genes had been successfully integrated into the genome of Y. lipolytica and the engineered strains ScDGAT2A, ScDGAT2B, ScDGAT2C, and ScDGAT3 were obtained.
In this work, the expression of ScDGAT2A, ScDGAT2B, and ScDGAT2C resulted in a 21.94%, 18.12%, and 38.58% decrease in DCW content compared to the CK strain, respectively, except for the DCW of the ScDGAT3 engineered strain, which was not significantly changed compared to the CK strain (Fig. 1b).In addition, compared to the CK strain, the expression of ScDGAT2A, ScDGAT2B, and ScDGAT3 resulted in a 63.26%, 70.14%, and 98.59% decrease in lipid content, respectively (Fig. 1b).In contrast, the expression of ScDGAT2C resulted in a 24.25% increase in lipid content compared to the CK strain (Fig. 1b), indicating that ScDGAT2C is closely associated with lipid synthesis, which is similar to the results reported previously.Expression of DGAT from Arabidopsis thaliana in Nannochoropsis oceanica resulted in an increase in total lipid content from 18.72% of wild-type strain to 24.49% [24].In another study, overexpression of ACC1 and DGA1 in Y. lipolytica, the lipid content of the engineered strain increased from 8.7% to 41%.Moreover, the lipid content of the engineered strain reached 61.7% after 120 h cultivation [25].
Subsequently, the fatty acid profiles of CK and the four engineered strains were analyzed.The expression of all four DGAT genes resulted in significant changes in the fatty acid profiles (Fig. 1c), which is consistent with the results previously reported in the overexpression of DGAT gene in Thalassiosira pseudonana and Phaeodactylum tricornutum [26,27].In this work, the significant changes in the fatty acid profiles were C16:0 and C18:2.The C16:0 ratio in total fatty acids of ScDGAT2A, ScD-GAT2B, ScDGAT2C, and ScDGAT3 strain decreased from 19.23 ± 0.55% of CK strain to 16.02 ± 0.76%, 16.16 ± 0.60%, 13.46 ± 0.43%, and 16.83 ± 0.80%, respectively.In contrast, the C18:2 ratio in total fatty acids of ScDGAT2A, ScDGAT2B, ScDGAT2C, and ScD-GAT3 strain increased from 12.38 ± 0.26% of CK strain to 21.83 ± 0.99%, 23.02 ± 0.40%, 22.59 ± 0.37%, and 22.06 ± 0.30%, respectively.Compared to the CK strain, the C16:1 content was decreased in the ScDGAT2A, ScDGAT2B, and ScDGAT3C strains, whereas increased in the ScDGAT2C strain.In addition, the C18:2 content in the ScDGAT2C strain increased by 131.18% compared to the CK strain (Fig. 1d), indicating that the expression of ScDGAT2C enhanced the accumulation of C18:2 in TG.
Compared to the CK strain, the UFAs and SFAs contents in ScDGAT2A, ScDGAT2B, and ScDGAT3 strains were significantly reduced (Fig. 1e).Although SFAs content in ScDGAT2C strain was also reduced, the UFAs content was significantly increased by 40.52% compared to the CK strain (Fig. 1e), which was similar to the results previously reported [24].These results suggest that the expression of ScDGAT2C improved the assembly of UFAs-TG in Y. lipolytica.

Engineering the enzyme activity of DGAT s
Previous studies have shown that several conserved amino acid motifs are essential for the high-level enzyme activity of DGAT , such as the "YFP" and "PH" motif in yeast DGAT2, which are also conserved in animals and higher plants [28].The replacement of "YFP" to "AAA" and His 193 to Ala 193 in Saccharomyces cerevisiae has been reported to result in an almost complete loss of enzyme activity [28].Protein sequence alignment revealed that only ScDGAT2C protein had a complete "YFP" motif, while the ScDGAT2A and ScDGAT2B proteins had "HFS" and "FFG" motifs, respectively (Additional file 1: Fig. S2b, Fig. 2a), which may be the reason why enzyme activity of ScDGAT2C was higher than ScDGAT2A and ScDGAT2B.Therefore, we boldly speculate that replacing the "HFS" motif in ScDGAT2A and the "FFG" motif in ScDGAT2B with the "YFP" motif through site-directed mutation may be able to enhance the enzyme activity to some extent and thus improve lipid accumulation (Fig. 2a).Then, using the ScDGAT2A gene as a template, primer pairs ScDGAT2A-F/ScDGAT2AM-R and ScDGAT2AM-F/ ScDGAT2A-R were used to amplify and obtain two fragments with the size of 1814 bp and 1057 bp, respectively, and the ScDGAT2AM gene was subsequently obtained by homologous recombination of the two fragments (Fig. 2b).Similarly, Using the ScDGAT2B gene as a template, primer pairs ScDGAT2B-F/ScDGAT2BM-R and ScDGAT2BM-F/ScDGAT2B-R were used to amplify and obtain two fragments with the size of 1855 bp and 790 bp, respectively, and then the ScDGAT2BM gene was obtained by homologous recombination of the two fragments (Fig. 2b).Gene sequencing results show that the "HFS" motif in ScDGAT2A and the "FFG" motif in ScD-GAT2B have been successfully replaced with the "YFP" motif (Fig. 2c).Subsequently, the ScDGAT2AM and ScD-GAT2BM genes were integrated into the intF3 loci of the Y. lipolytica mutant (CK) strain genome in the same manner as described above and the ScDGAT2AM and ScDGAT2BM strains were obtained.
Compared to the ScDGAT2A strain, the SFAs content of ScDGAT2AM strain showed no significant change, while the UFAs content was significantly increased by 22.83%.Compared to the ScDGAT2B strain, the ScD-GAT2BM strain showed a significant decrease in SFAs content by 31.13%, while the UFAs content was significantly increased by 11.55% (Fig. 2g).These results indicate that the replacement of the "HFS" motif in ScDGAT2A and the "FFG" motif in ScDGAT2B with the "YFP" motif enhanced the assembly of UFAs-TG in Y. lipolytica.

Effects of DGAT s on lipid and EPA accumulation
We explored the "push-pull-promote" strategy to enhance EPA synthesis.First, more EPA was pulled into the TG for storage by expression of DGAT s (pull strategy).According to reports, DGAT2 plays an important role in introducing unusual fatty acids such as PUFAs into lipids and TG [29].Due to the desaturase/elongase pathway in Y. lipolytica is incomplete, naturally producing mainly C16:0, C16:1, C18:0, C18:1, and C18:2.Therefore, to further evaluate the effect of DGAT on the accumulation of PUFAs, a Y. lipolytica mutant strain (named yl-EPA) capable of producing EPA was used, which had been obtained in our laboratory by transferring Δ12 desaturase, Δ9 elongase, Δ8 desaturase, Δ5 desaturase, and Δ17 desaturase into PO1f (Table 1).Subsequently, the ScDGAT2C, ScDGAT2AM, and ScDGAT2BM genes were integrated into the intF3 loci of the yl-EPA strain in the same manner as described above to investigate the effect of DGAT on PUFAs synthesis (Fig. 3a).As seen in Additional file 1: Fig. S6b, these DGAT genes had been successfully integrated into the genome of Y. lipolytica and the engineered strains yl-EPA::ScDGAT2C, yl-EPA::ScDGAT2AM, and yl-EPA::ScDGAT2BM were obtained.
In addition, the EPA content and titer of yl-EPA::ScDGAT2AM and yl-EPA::ScDGAT2BM strains were not significantly different compared to the yl-EPA strain, whereas the EPA content and titer of yl-EPA::ScDGAT2C strain were increased by 135.42% and 108.57%, respectively (Fig. 3e), which may be due to the weaker activity of ScDGAT2AM and ScDGAT2BM in transferring the acyl portion of EPA-CoA to DG for TG synthesis, resulting in the release of EPA from polar lipids that were not well incorporated into TG for storage and was instead transported to the β-oxidation pathway for degradation [8].In contrast, the expression of ScDGAT2C provides a strong pulling power to mediate the incorporation of EPA into TG for storage and protection, resulting in the enrichment of EPA in TG and the increased level of TG-derived EPA.It has been reported that the expression of CrDGTT1 from Chromochloris zoftiensis in Nannochoropsis oceanica increased the percentage of EPA in TG by 4.8-fold compared to the control strain.In addition, the EPA content derived from TG reached 10.3 mg/g DCW, which was 10.1-fold higher than of the control strain [8].In addition to EPA, we also found that the expression of ScDGAT2C, ScDGAT2AM, and ScDGAT2BM genes resulted in an increase in C18:2, C18:3, C20:2, and C20:4 titer (Additional file 1: Fig. S8), suggesting that ScDGAT2C, ScDGAT2AM, and ScD-GAT2BM may play an important role in incorporating these PUFAs into TG storage.Therefore, the application of these DGAT genes for metabolic engineering modification of other PUFAs synthesis could be considered in the future.

Lipomics analysis of the effect of ScDGAT2C on EPA storage
Triglyceride/fatty acid (TG/FA) cycling is the process of partial or complete degradation of stored fat to release free FAs that subsequently are used to resynthesize a new molecule of TG [30].According to reports, some marine microalgae such as N. oceanica and P. tricornutom can synthesize EPA, but it is mainly stored in polar membrane lipids rather than in lipid TG, which may be the reason for its limited content at a low level [8].Unlike membrane lipids, TG is stored in lipid droplets and has the potential for high-level accumulation in microorganisms.In addition, the physiological properties and function of TG molecules depend on the composition of fatty acids attached to their glycerol backbone, which further highlights the potential application prospects of TG molecules.Therefore, channeling EPA to TG may be a promising way to overcome EPA storage limitations.Therefore, in order to further investigate the effect of ScDGAT2C on EPA storage in TG, lipidomics analysis was performed on yl-EPA and yl-EPA::ScDGAT2C strain.The experimental procedures included lipid extraction, LC/MS analysis, data acquisition, and bioinformatics analysis (Fig. 4a).
(See figure on next page.)Fig. 4 Lipomics analysis of the effect of ScDGAT 2C on EPA storage.a Schematic of the steps in the workflow of lipomics analysis.b Effects of ScDGAT 2C on glycerolipids, glycerophospholipids, sphingolipids, diacylglycerol (DG), and Triglyceride (TG) in yl-EPA strain.c Volcano plot analysis of yl-EPA::ScDGAT2C and yl-EPA.Red represents up-regulated metabolites, blue represents down-regulated metabolites, and gray represents unchanged metabolites.d Effects of ScDGAT 2C on EPA-related TG in yl-EPA strain.e Effects of ScDGAT 2C on EPA-related DG, phosphatidylcholine (PC), Bis-methyl phosphatidic acid (BisMePA), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) in yl-EPA strain.(f) Clustering analysis of lipid metabolites with the top 50 variable importance (VIP) values under ScDGAT 2C expression conditions.Each row of the color block gradient map on the left represents a metabolite, and the different colors represent the relative abundance of metabolites in the sample set.The right side is the VIP bar graph of metabolites, the length of the bar represents the contribution of the metabolite to the difference between two groups and the color of the bar indicates the significance of the difference.Three biological replicates were used and mean values ± SD (n = 3) are shown.Two-way ANOVA with Tukey's multiple comparisons test.*P < 0.05, **P < 0.01, and ***p < 0.001 compared with the yl-EPA Glycerolipids, glycerophospholipids, and sphingolipids are the main components of lipid metabolites.As seen in Fig. 4b, the expression of ScDGAT2C resulted in a decrease in the relative content of glycerophospholipids from 61.83% of yl-EPA to 34.47%.In contrast, the relative contents of sphingolipids and glycerolipids increased from 32.81% and 5.36% of yl-EPA to 57.39% and 8.14%, respectively.In addition, the expression of ScDGAT2C resulted in an increase in the relative content of TG from 79.43% of yl-EPA to 81.32%, indicating that the expression of ScDGAT2C promoted the biosynthesis of TG.

GPATs further enhanced the storage of EPA in TG
Due to its low activity and position as a first step, glycerol-3-phosphate acyltransferase (GPAT) is considered a rate-limiting enzyme in TG biosynthesis.Strategies to increase lipid accumulation by enhancing GPAT expression have been successfully applied in a variety of microalgal species [14].According to reports, blocking GPAT expression leads to a decrease in lipid accumulation, suggesting its crucial role in TG formation [31].The expression of GPAT gene in Phaeodactylum triornuum not only promoted oil body formation, but also stimulated a double increase in neutral lipid content [32].Therefore, on the basis of the yl-EPA::ScDGAT2C strain, GPATs was further introduced to pull more EPA into the TG for storage (pull strategy).(Fig. 5a).
Based on functional annotation of the genome of Schizochytrium sp.HX-308 and the phylogenetic analysis of amino acid sequences, two GPAT genes, ScGPAT1 (gene ID: A1558) and ScGPAT2 (gene ID: A5476), were identified (Fig. 5b).Multiple-sequence alignment of Schizochytrium sp.HX-308 GPATs was performed with other GPAT amino acid sequences.ScGPAT1 and ScGPAT2 were found to contain four highly conserved motifs that have been suggested to play a role in the activities of acyltransferases (Additional file 1: Fig. S11) [33].In this work, compared to the yl-EPA::ScDGAT2C strain, the expression of ScG-PAT1 and ScGPAT2 genes increased lipid content by 21.92% and 28.92%, respectively (Fig. 5c).According to reports, heterologous overexpression of GPAT from Lophosphaera incise in C. reinhardtii increased TG content to 50% of DCW without growth restriction [34].Compared to the yl-EPA::ScDGAT2C strain, the fatty acid profiles in yl-EPA::ScDGAT2C::ScGPAT1 and yl-EPA::ScDGAT2C::ScGPAT2 strains did not show significant changes (Fig. 5d).However, the expression of ScGPAT1 and ScGPAT2 resulted in an increase of 28.38% and 38.30% in PUFAs content, and an increase of 17.83% and 29.59% in SFAs content, respectively (Fig. 5e).In addition, compared to the yl-EPA::ScDGAT2C strain, the expression of ScGPAT1 resulted in a 29.68% and 15.32% increase in EPA content and titer, respectively, and the expression of ScGPAT2 resulted in a 37.91% and 23.00% increase in EPA content and titer, respectively (Fig. 5f ).

Enhancing the precursor and cofactor supply to improve EPA accumulation
Notably, in this study, although DGATs and GPATs provide pull to incorporate more EPA into TG storage, EPA was still below satisfactory levels due to the limitations of their lipid content [35].Therefore, we further increase precursor and cofactor supply to enhance the synthesis of EPA and lipids.Acetyl-CoA is the actual form in which glycolytic pyruvate enters the tricarboxylic acid (TCA) cycle, as well as being an important precursor for fatty acid synthesis [36].Acetyl-CoA synthetase (ACS) is an enzyme involved in carbon metabolism, which converts acetic acid into acetyl-CoA in an irreversible reaction [37].It was reported that when the ACS gene from Thalassiosira pseudonana was heterologously expressed in the Saccharomyces cerevisiae fatty acid activation deletion strain (faa4D), the EPA level of the engineered strain was increased by more than 75-fold compared to the control [38].Pyruvate dehydrogenase complex (PDC) connects the carbon flux from glycolysis with the tricarboxylic acid cycle by converting pyruvate into acetyl-CoA [39].Acetyl-CoA from the cytoplasm is converted into malonyl-CoA by acetyl-CoA carboxylase (ACC ).Subsequently, acetyl-CoA is used as the starting compound and malonyl-CoA as the elongation unit to continuously elongate fatty acids by C2 units until C16:0 or C18:0 acyl-CoA is synthesized via a type I fatty acid synthase (FAS).Besides, in addition to the initial two-carbon skeletons, fatty acid synthesis requires two NADPH molecules to extend the saturated C2 unit, as well as each double bond generated on the existing carbon skeleton [40].Therefore, enhancing the supply of NADPH is not only beneficial for improving lipid synthesis but also for the synthesis of very long-chain PUFAs [41].Glucose 6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway (PPP) that provides reducing power in the form of NADPH [22].Based on functional annotation of the genome of Schizochytrium sp.HX-308 and the phylogenetic analysis of amino acid sequences, ScACC (gene ID: A2520), ScACS (gene ID: A2978), ScPDC (gene ID: A8969), and ScG6PD (gene ID: A6492) were identified (Fig. 6a).Therefore, based on the yl-EPA::ScDGAT2C::ScGPAT2 (named yl-EPA-1) strain, the supply of precursors required for lipid and EPA synthesis was enhanced by expression of ScACC , ScACS, and ScPDC (push strategy), as well as the supply of NADPH required for lipid and EPA synthesis was enhanced by expression of ScG6PD (promote strategy) (Fig. 6b).

Conclusions
In this study, we explored the lipid synthesis genes of Schizochytrium sp. and constructed a Y. lipolytica cell factory through a rationally designed "push-pull-promote"

Strains and media
All the strains and plasmids employed in this work are listed in Additional file 1: Table S2.Schizochytrium sp.HX-308 (CCTCC M209059) strain used in this work has been preserved in the China Center for Type Culture Collection (CCTCC) [44].Escherichia coli DH5α was cultured in Luria-Bertani (LB) medium with ampicillin at 37 ℃ and 200 rpm.All Y. lipolytica transformants were grown in yeast peptone dextrose (YPD) medium at 30 ℃ and 220 rpm.The appropriate yeast transformants were isolated by using synthetic complete medium without uracil (SC-Ura) plates.The URA3 maker was recycled by selection on YPD plates supplemented with 1 g/L 5-fluoroorotic acid (5-FOA) and 15 g/L agar.The medium and conditions required for the fermentation of Y. lipolytica in this work were also described previously [45].
Then, when the OD 600 of the strain to be transformed cultured in YPD medium reached between 0.8 and 1.0, the Y. lipolytica cells were centrifuged for transformation.The recombinant plasmids were transferred into Y. lipolytica by using the Frozen-EZ Yeast Transformation II Kit (Zymo Research, Orange, CA) according to the manufacturer's instructions.The appropriate yeast transformants were isolated by using SC-Ura plates and the DNA of these transformants was isolated using an E.Z.N.A. ® Yeast DNA Kit (Omega Bio-Tek Inc., USA) and verified by PCR.The reagents and methods used for plasmid construction and transformation in this work were also described previously [45].The modified DNA fragments and plasmids were sequenced by Sangon Biotech Co., Ltd.(Shanghai, China).The recombinant strain was incubated on YPD + 5-FOA plates for 2-3 days to remove the URA3 counter-selection marker, as strains expressing URA3 did not grow in the presence of 5-FOA.All the primers used for the genetic modification in this work are listed in Additional file 1: Table S3.

DCW, total lipid, and fatty acid analysis
The methods of DCW determination, lipid extraction, and fatty acid methyl esters (FAMEs) in this work were described previously [45].After the fermentation, 40 mL of the fermentation broth was centrifuged and washed twice with water, and then lyophilized for 48 h for DCW quantification.About 2 mg of lyophilized cell were weighed and 500 μL 1 M sodium hydroxide in methanol with 100 μL of heptadecanoic acid (C17:0) was added and shaken at 1200 rpm for 2 h.Subsequently, 40 μL of 98% sulfuric acid was added, followed by 400 μL of hexane and shaken at 1200 rpm for 10 min to extract fatty acid methyl ester (FAME).After centrifugation at 8000 rpm for 2 min, the top hexane layer was taken for GC analysis.The prepared FAMEs samples were analyzed by a GC-2030 gas chromatography system (GC-2030, Shimadzu, Japan) equipped with a flame ion detector (FID) and a DB-23 GC column (60.0 m × 0.25 mm × 0.25 μm, Agilent, USA).

Lipidomics analysis
At the end of fermentation, samples were collected and centrifuged at 4 °C and 5000 g for 5 min, during which time they were quickly rinsed 2-3 times with pre-cooled PBS solution.The samples were then stored at -80 °C for subsequent lipidomics analysis.
The 50 mg sample was weighed into a 2 mL centrifuge tube, to which a 6 mm diameter grinding bead, 200 μL of extraction solution (isopropanol: acetonitrile = 1:1), and 400 μL of MTBE were added and placed in a frozen tissue grinder at − 10 °C for 6 min, followed by ultrasonic extraction at 5 °C for 30 min, and then stand at − 20 °C for 30 min.Next, the samples were centrifuged at 4 °C and 13000g for 15 min, 350 μL of supernatant was taken into a centrifuge tube and blown dry with nitrogen.Then, the samples were re-dissolve by adding 100 μL of extraction solution, followed by vortexing for 30 s and then ultrasonic extraction at 5 °C for 5 min.Subsequently, it was centrifuged at 4 °C and 13000g for 10 min and the supernatant was removed for lipidomics analysis.Lipidomics analysis was performed by Shanghai Majorbio Biopharm Biotechnology Co., Ltd.(Shanghai, China).

Statistical analysis
Three independent replicates were performed for all experiments, with results presented as mean ± standard deviation (SD).Student's t test and Two-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis.P values of < 0.05 were considered statistically significant, and statistical significance is indicated as *P < 0.05, **P < 0.01, and ***p < 0.001.

Fig. 1
Fig. 1 Effects of DGAT s on lipid and fatty acid synthesis in Y. lipolytica mutant strain (CK).a Scheme of the expression of DGAT genes controlled by the TEFin promoter and CYC1t terminator at the intF3 integration site based on homologous recombination.intF3-Up, upstream homologous arm of integration site intF3; intF3-Down, downstream homologous arm of integration site intF3.Effects of DGAT s on lipid and DCW(b), fatty acid composition (c), C16:0, C16:1, and C18:2 content (d), and UFAs and SFAs content (e) in Y. lipolytica mutant strain (CK).Three biological replicates were used and mean values ± SD (n = 3) are shown.Two-way ANOVA with Tukey's multiple comparisons test.*P < 0.05, **P < 0.01, and ***p < 0.001 compared with the CK

(
See figure on next page.)Fig. 2 Effect of site-directed mutation of DGAT s on lipid and EPA synthesis.(a) The region of site-directed mutation in ScDGAT2A and ScDGAT2B.(b) Schematic diagram of site-directed mutation of ScDGAT2A and ScDGAT2B.(c) Sanger sequencing confirmation of site-directed mutation of ScDGAT2A and ScDGAT2B.Effects of site-directed mutation of ScDGAT2A and ScDGAT2B on lipid and DCW (d), fatty acid composition (e), C16:0 and C18:2 content (f), and UFAs and SFAs content (g) in Y. lipolytica mutant strain (CK).Three biological replicates were used and mean values ± SD (n = 3) are shown.Student's t test was used for statistical analysis, and statistical significance is indicated as *P < 0.05, **P < 0.01, and ***p < 0.001